1. Field of the Invention
Immunofluorescence techniques are used for the determination of the presence of a material of interest (analyte) in a specimen (sample). Many materials of interest are present in biological specimens, for example, on a macromolecular surface such as a cell, DNA probe, etc. Specific immunofluorescence techniques involve fluorescent antibody staining. Generally, there are three techniques available: the direct method, the indirect method; and the complement staining method.
In the direct technique, a specific antiserum for an agent, which is generally the material of interest, is prepared in a laboratory animal, or a monoclonal antibody specific for the agent is prepared. The protein or more often the gamma globulin fraction of the serum, is labeled with a fluorescing compound, usually a fluorescein derivative. The antibodies against the selected agent, labeled with fluorescein, are then used to detect that agent in a specimen or tissue culture. A slide preparation of the specimen is treated with the fluorescein-labeled antiserum conjugate under suitable conditions, and, if the homologous antigen is present, an antigen-antibody reaction will take place. The specimen is then washed free of non-specifically bound conjugate. Areas of the preparation exhibiting fluorescence under the fluorescence microscope or instrument such as a solid-reading fluorometer indicate the presence of homologous antigen, provided adequate control procedures have been carried out to insure that the binding is specific.
In the indirect fluorescent antibody technique unlabeled antibody specific for a particular antigen is combined with the antigen thus giving an antigen-antibody complex. Anti-antibody (antibody for antibody) is produced according to conventional procedures and labeled witn a fluorescing compound. This conjugate combines with the antibody portion of the antigen-antibody complex produced above if the antigen is present in a sample to be analyzed. After appropriate washing, the sample is examined for the presence of fluorescence.
In the complement staining method the sample containing the material of interest is treated with unlabeled antibody specific for a particular antigen and then with a mixture of inactivated unlabeled antiserum and complement and then rinsed. This is followed by the application of, e.g., fluorescein-conjugated anti-(guinea pig complement) which binds to the site of any complement fixing antigen-antibody reaction. Fluorescent areas in the specimen indicate the sites where antigen is present.
One problem confronting users of the above-described techniques is non-specific fluorescence which tends to reduce or eliminate the effectiveness of the particular test. Generally, the specimen to be analyzed contains tissue, cells, microorganisms such as bacteria and fungi, cellular constituents, and debris. In many cases, the presence of these materials gives rise to non-specific staining (non-specific fluorescence) in fluorescent antibody techniques leading to false positive or false negative results. Those skilled in the art believed the non-specific effect to be caused primarily (1) by charge interaction between proteins in the specimen to be analyzed and proteins employed in the conjugate, (2) by other physio-chemical interactions between the proteins contributing to the non-specific staining, for example, hydrogen bonding and physical entrapment and (3) by non-specific immuno-reactions. The problem of non-specific staining is severe in virus antigen detection and the detection of bacteria or fungi.
As exemplary of the problems encountered with viruses are those problems encountered with members of the herpes virus group. Specimens to be examined for infection contain not only specific cells but also cellular debris, bacteria, and fungi. The presence of these materials gives rise to non-specific fluorescence which can mask the specific fluorescence. As a result, accuracy of the test is diminished or lost.
Various methods have been advanced for controlling non-specific staining in immunofluorescent techniques, for example, dilution of antisera; control of pH; adsorption of antisera with tissue preparations; column purification of antisera; counterstaining with a dye which contrasts with the specific dye, e.g. Evan's blue; and, with fluorescein as the fluorescent agent, using a non-specific dye such as rhodamine or a conjugate of rhodamine and a protein. Rhodamine has absorption maxima at about 515 and 552 nm and has a positive charge. None of the above methods has proven to be effective for all immunofluorescent techniques.
2. Description of the Prior Art
Applications of immunofluorescence are described by Gardner, et al., "Rapid Virus Diagnosis", Butterworth (Publishers) Inc., Boston, Massachusetts (1980) and by Goldman, "Fluorescent Antibody Methods", Academic Press, New York, N.Y. (1968). Use of a contrasting fluorescent dye (rhodamine-conjugated normal serum) as a counterstain in fixed tissue preparations is described by Smith, et al., Proceedings of the Society of Experimental Biology and Medicine, 102, 179-181 (1959). Rhodamine-conjugated papain as a contrasting dye in fluorescence microscopy is discussed by Alexander, et al., Immunology 6, 450-452 (1963). A novel dipole-dipole coupled fluorescence energy transfer acceptor, 4',5'-dimethoxy-6-carboxy-fluorescein, useful for fluorescence immunoassays is discussed by Khanna, et al., Analytical Biochemistry, 108, 156-161 (1980). In U.S. Pat. Nos. 4,318,846 and 4,351,760 there are disclosed fluorescein-poly(amino acid) conjugates as fluorescers and quenchers.